Current microbial tests are qualitative which pose definite problems. Two notable problems are the slowness of achieving results, and the high cost and availability of certain reagents used in various test formulations.
In recent years, the use of chromogenic, fluorogenic and non-chromogenic enzyme indicators has come into increasing use due to ease of use and rapid detection, especially in the field of microbiology. Edberg patented (U.S. Pat. No. 4,925,789) the use of one soluble fluorogen (methyumbelliferone-fluorescent blue) for detection of beta-galactosidase and one soluble chromogen (nitrophenol-yellow) for the detection of beta glucuronidase. This test is in a broth. However, literature (see Brenner U.S. Pat. Nos. 6,063,590; 6,306,621; and 6,670,145) has taught that fluorogens and chromogens which yield soluble products are not good for use on traditional agar or other gel type plate tests or for membrane filtration tests because the soluble indicator does not stay with the microbial colony making it easy to detect, but spreads out across the plate so one can not tell what it is (or is not) supposed to indicate. One answer to this problem came with the discovery that indolyl substrates, especially halogenated indolyl substrates, can be enzymatically cleaved to produce insoluble colors that will stay with a microbial colony, and make its detection simple. Ley (U.S. Pat. No. 4,923,804) took advantage of this idea and patented the use of indolyl beta glucuronidase to detect E. coli with membrane filtration. Roth (U.S. Pat. No. 5,210,022) was the first to use two colors of insoluble chromogens to detect two different enzymes at once, and put it into practice. Ferguson (U.S. Pat. No. 5,358,854) synthesized the novel chloroindolyl galactoside for Roth to detect coliforms, and Roth combined this with bromo-chloro-glucuronide to detect and differentiate E. coli and other coliforms simultaneously. Another proposed approach used a soluble fluorogen and an insoluble chromogen, but the use of soluble fluorogenic and/or chromogenic substrates in a solid gel or membrane filtration test has been seen as undesirable, again, due to the diffusible nature of their products (see Brenner patents).